Vol. 171, No. 3, 1990 September

BIOCHEMICAL

28, 1990

GLYCOSYLATION

OF THE AMYLOID

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1015-1021

PEI’TIDE PRECURSOR CONTAINING THE

KUNITZ PROTEASE INHIBITOR DOMAIN IMPROVES TIiE INHIBITION OF TRYPSIN Edmond Godfroid and Jean-Noel Octave* Universite Catholique de Louvain, Laboratoire de Neurochimie, UCL 1352, Ave Hippocrate 10, B-1200 BRUXELLES, Belgium Received

August

6, 1990

The amyloid S peptide (ABP) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer’s disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain. 01990Academic press,rnc.

The first cDNA

library

full-length indicated

cDNA sequence isolated from a foetal that

human brain

the APP is a 695 amino acid transmembrane

protein (APP 695), with a large extracellular amino terminal portion and a short carboxyl-terminal tail in the cytoplasm (1). The AI3P consists of 28 amino acids outside the membrane and 11 to

14 amino acids in the hydrophobic

transmemhrane domain. There are alternatively

spliced APP transcripts and the

two major corresponding proteins (APP 751 and APP 770) contain inserts which are homologous to the Kunitz serine protease inhibitors (2,3,4). Amyloid

peptide precursors are not only transmembrane proteins, but are

also found in cell culture medium as well as in cerebra-spinal fluid (5,6). These soluble proteins are recognized

by anti-amino-terminal antibodies but not by anti-carboxyl-terminal antibodies, indicating that the soluble forms are obtained by proteolytic cleavage of the transmembrane precursors. The soluble APPs found in the cerebra-spinal fluid are recognized by anti-amyloid

B

* To whom correspondence should be addressed.

1015

ooO6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

171,

No.

peptide

3, 1990

antibodies

fected

cells,

peptide

(8).

end,

BIOCHEMICAL

(7), and very

the

precursor

AND

BIOPHYSICAL

recently,

RESEARCH

it was

is cleaved

COMMUNICATIONS

demonstrated

at position

that,

15 within

the

in transamyloid

6

We have isolated an additional transcript of the APP gene (9). At the 5’ the cDNA sequence is identical to the Kang sequence. The sequence

contains

the KPI domain,

but the 208 amino

acids at the carboxyl-terminus

the protein, including the ABP, are replaced by 20 non hydrophobic leading to a 563 amino acid protein (APP 563). The corresponding is much less abundant

than the 3.2, 3.4 kb mRNA

the divergence

at a splicing

divergent

occurs

sequence

as a probe,

region of the chromosome The secreted

full-length

vector. After transfection, medium in immunobloting been recently bacterial

fusion

reported product,

At the DNA

level,

transcript

was

mapped

to the same

the APP gene is found.

sequences

encoding

the KPI domain the cells experiment (10)

amino acids 2 kb mRNA

at the end of exon 11, and using the

the new

21 where

cDNA

APP containing

site

doublet.

of

that

the

transmembrane

and

cloned

in the pKCR3

tansfection

were analysed and in reverse the

is sufficient

were

Kunitz for

trypsin

together with enzymography.

sequence, inhibition

even

their culture While it has

expressed

in vitro,

the

as a

our results

indicate that the glycosylation of the APPs containing the KPI domain improves the inhibition of trypsin detected by reverse enzymography.

greatly

METHODS Cell culture and transfection. Cos-1 cells were grown at 37°C under humidified atmosphere and 10 % CO2 in Dubelcco’s modified Eagles medium (DMEM) containing lpgr/ml biotin, 1 % non-essential amino acids, and 5 % fetal calf serum. The semi-confluent cells cultured in 63 cm2 dishes were transiently transfected with 10 pgr of recombinant or non recombinant plasmid DNA using the DEAE dextran as a carrier molecule allowing the phagocytosis of the DNA. The transfected cells were incubated for 2 hours at 37°C with DMEM supplemented with 5 % fetal calf serum and 100 PM chloroquine. After two washing steps with prewarmed serum-free DMEM, transfected cells were incubated for 48 hours at 37°C with DMEM containing 5 % fetal calf serum. The culture was then treated for 24 hours at 37°C with serum-free DMEM supplemented with 5 &ml insulin. Cells were harvested together with their culture medium and analyzed in immunoblotting and reverse enzymography experiments. Production of monoclonal antibodies against APP. -To generate antibodies against the extracellular nortion of the APP containing the KPI domain. a Pstl fragment (positions 240-1383 in the Kang sequence) ias cloned in the’ pUEX-1 bacterial expression vector downstream the lac Z gene sequence. After transformation, bacteria produced a fusion product with B-galactosidase which was purified and used for production of monoclonal antibodies. SDS-PAGE and immunoblottina. Transfected cells overexpressing the transmembrane or the secreted APP were analysed together with their culture medium on SDS-PAGE containing 7.5 % polyacrylamide. Electrophoresis was carried out for 2 hours at 140 volts. The immunoblotting was performed by electroblotting the proteins from the polyacrylamide gel onto nitrocellulose membrane. The

Vol.

171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

filter was blocked with 5 % non-fat dry milk in TBST (Tris 50 mM pH 8.1 / NaCl 150 mM / Tween 20 0.5 %I, incubated at room temperature for 16 hours with primary antibody and finally immunostained with alkaline phosphatase labeled second antibody according to the procedures suggested by the manufacturer (Promega). The monoclonal antibody was produced in ascite fluid used at a dilution of l/1,000. SDS-PAGE and reverse enzvmoaraDhv. Proteins from conditionned medium or cell extracts were subjected without prior boiling to SDS-PAGE in 7.5 % polyacrylamide gels under non-reducing conditions. Electrophoresis was carried out for 2 hours at 140 volts. The gels were rinsed 3 times for 45 minutes in 2.5 % Triton X-100 and 3 times for 10 minutes in 0.1 M Tris, pH 8.1. The gels were then layered on 0.8 % agar gels containing 15 mg/ml non-fat dry milk solubilized in 0.1 M Tris-HCl buffer pH 8.1 and 100 ngr/ml Trypsin. During a 10 to 12 hours incubation at 37”C, the proteins diffused from the gel to the agar layer. Trypsin was able to digest and clarify casein but not where inhibition of trypsin occured. Chemical dePlvcosvlation. The proteins recovered in the culture medium of transfected cells were treated by trifluoromethanesulfonic acid according to the method described by Edge et al. (11). The deglycosylated proteins were analyzed in immunoblotting experiments. Enzvmatic deglvcosvlation. Proteins recovered in the conditionned medium of cells transfected with the sequence encoding the secreted APP were deglycosylated by treatment with neuraminidase or with N-glycosidase F and Endo-a-Nacetylgalactosaminidase (O-glycan-peptide hydrolase). All these enzymes were provided from Boehringer Mannheim Biochemica (FRG). For the neuraminidase treatment., lyophylised culture medium was incubated with 1.5 mU enzyme in 50 mM sodium acetate buffer (pH 5.0) for 2 hours at 37°C. The reaction was stopped by addition of non-reducing Laemmli buffer (12). For the N- and Oglycosidase treatment, lyophylised culture medium was incubated with 0.4 U of N-glycosidase F and 1 mU Endo-a-N-acetylgalactosaminidase in 50 mM sodium phosphate buffer (pH 7.1) containing 25 mM EDTA and 0.1 % SDS for 18 hours at 37°C. 10 % n-octylglucoside was added‘ before adding N-glycosidase-F in order to avoid inactivation of the enzyme by SDS. Control experiments were performed in the same buffers and for the same incubation periods but without enzymes. FUZSULTSAND DIscUSSION COS-1

cells

were

transiently

transfected

with

the

cDNA

sequences

encoding the transmembrane or the secreted APP and analysed together with their culture medium in immublotting experiments. The results presented in figure 1 indicate that after transfection

of the cells with the non recombinant

plasmid, the monoclonal antibody did not detect

any proteins. When the cells

were transfected with the sequence encoding the transmembrane or the secreted protein, the antibody detected the expression of APP 770 and APP 563 respectively. Despite the fact that the soluble APP 770 is obtained by proteolytic cleavage of the transmembrane precursor, the protein is found in the culture medium at a molecular weight similar to that of the cell associated protein. The secreted APP 563 has not to be cleaved for secretion and the protein in the culture medium has a higher molecular weight than the cell associated protein (Figure 1 and 2 A). 1017

Vol.

171, No. 3, 1990

BIOCHEMICAL

cos -1 CELLS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CULTURE MEDIUM

-II 0

Kd 205 *

a

E a

I

ii ua

E I-

a

a

I

I = E ii”

I: iii

I: VJ I ti “kS

5 E

116 *

01

77 *

Kd 116 *

46.5 *

77 -

02

= B

A

Figure 1 : COS-1 cells were transfected with the pKCR3 non recombinant plasmid f-1 or the same vector containing the sequenceencoding the APP 563 (A 563) or the APP 770 (A770). Cells and culture medium were analyzed in immunoblottingexperiments as describedin methods. Figure 2 : COS-1 cells were transfected with the sequenceencoding the APP 563 and analyzed together with their culture medium in immunoblotting experiments. In panel B, the proteins recovered in the culture medium were deglycosylatedusing the trifluoromethanesulfonicacid (TFMS). The amyloid peptide precursors are N- and 0-glycosylated translational modifications weight concomitant with

could be responsible for

(5). These post

an increase in molecular

way, the protein the secretion process. In that recovered in the culture medium was chemically deglycosylated using trifluoro methanesulfonic acid. After the secreted protein medium and cell enzymatically

this treatment

(Figure 2B) the molecular weight of

decreased but remained at a middle distance between

associated protein.

deglycosylated

When the

extracellular

using neuraminidase toghether

proteins

with

were

N- and O-

glycosidases (Figure 4 A), the molecular weight of the soluble APP was decreased but remained higher than that of the cell associated protein. This could be the result of uncomplete deglycosylation or may be that postranslational modifications other than glycosylation are involved in the shift of molecular weight. This later hypothesis is futher sustained by the fact that when both the medium and cell associated proteins are enzymatically

deglyco-

sylated (data not shown), the shift in molecular weight still persists. It has been previously described by Kitaguchi and colleagues (41 that the amyloid peptide precursors containing the Kunitz domain are able to inhibit trypsin. We have studied trypsin inhibition using reverse enzymography. COS cells were transfected and analyzed together with their culture medium in immunoblotting experiments and reverse enzymography. After transfection of the non recombinant plasmid (figure 3A), an endogenous expression of the APP 770 was observed. APP 770 and APPP 563 were overexpressed by transfection

Vol. 171, No. 3, 1990

BIOCHEMICAL

A770

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A563

-nCMCMCM

Kd 116 -

0

04 Figure 3 : COS-1 cells were transfected with the non recombinant plasmid (-) or the plasmid containing the sequence encoding the APP 770 (A770) or the APP 563 (A563). Cells (C) and culture medium (M) were analyzed in immunobloting (A) and reverse enzymography (8) experiments. Figure 4 : COS-1 cells were transfected with the sequence encoding the APP 563 and analyzed with their culture medium in immunobloting (A) or reverse enzymography (B) experiments. Proteins corresponding to the endogenous APP 770 (Q) and the transfected APP 563 (a) were deglycosylated using neuraminidase (NEURAM.) and N- and 0-glycosydases (N + 0 GLYC.). of

the

corresponding

enzyrnographic while

technique

the

inhibition

sequences.

antibody

and

stains

The the

proteins

same

results

proteins

obtained

both

in

cells

were in

analyzed

figure and

3B

using indicate

the that

medium, the

culture

of trypsin only occurs in the culture medium at a molecular weight

of soluble proteins overexpressed by transfection

of the corresponding cDNA

sequences. Since

the

difference

in

molecular

weight

between

cells

and

culture

medium is related to post-translational modifications including glycosylation, we have tried to see whether these post-translational modifications were important for inhibition of trypsin. The soluble proteins of the culture medium corresponding to endogenous expression of deglycosylated by treatment

APP

770

and

transfected

APP

563

were

with neuraminidase or with N- and 0-glycosidases.

After these treatments, 30 % of the proteins were used for detection by the antibody (Figure 4A) and the remaining 70 % were analyzed in reverse enzyrnography

rating

(Figure

4B).

After

treatment

condition at pH 5, the inhibition

with

neuraminidase

in

non

denatu-

of trypsin by APP 770 seems to be

the inhibition by APP 563 is conserved. However, after treatreduced, while ment with N- and 0-glycosidases at pH 7, trypsin inhibition is abolished for both APP 770 and APP 563.

1019

Vol.

171,

No.

3, 1990

BIOCHEMICAL

In conclusion,

overexpression

transient

transfection

recovered

in the culture

cell associated This is related

of

COS

of

different

cells

medium

BIOPHYSICAL

RESEARCH

amyloid

indicate

that

at a molecular

the

weight

COMMUNICATIONS

peptide

precursors

by

secreted

proteins

are

higher than that of the

proteins.

increase

in molecular

to several

Inhibition measured

AND

post-translational

of

trypsin

by several

these very sensitive the

by

methods,

sequence

of

as a bacterial

APP

the

APP

glycosylation.

the

domain

containing

it was recently

homologous

to

the secretion

including

using chromogenic

fusion product

substrates

might

observed

between

in viva,

the control

precursor efficient

that

with

KPI

substrates

reported

the

has been

(4,10,13).

that

Kunitz

is sufficient

process

the

Using

amino

protease

inhibitor

for trypsin

inhibition

acid

domain, (10).

of APP 770 and 563 overexpressed greatly improves the by transfection of the corresponding cDNA sequences inhibition of trypsin detected by reverse enzymography. The use of chromogenic

here

concomitant

modifications

investigators

expressed

We report

weight

the glycosylation

be

medium

too

sensitive

to

and cell associated

of the activity

make proteins.

of brain proteases

the

difference

Our results

we

have

indicate

that

by the amyloid

peptide

containing the Kunitz protease inhibitor domain could be much more if the protein goes through post-translational modifications including

glycosylation. ACKNOWLJZDGh4ENTS We thank F. de Sauvage and J. Limet for antibodies, P. Leprince for interesting discussion

the production of monoclonal related to enzymography and

B. Tasiaux-Doumont for excellent technical assistance. by the belgian F.N.R.S and the Queen Elisabeth Medical

This work was supported Foundation.

REFERENCES 1.

Kang J., Lemaire H.-G., Unterbeck A., Salbaum J.M., Masters Grzeschik K.-H., Multhaup G., Beyreuther K., and Miiller-Hill Nature 325, 733-736.

2.

Ponte P., Gonzalez-DeWhitt P., Schilling J., Miller J., Hsu D., Greenberg D., Davis K., Wallace W., Lieberburg I., Fuller F., and Cordell B. (1988) Nature 331, 525-527.

3.

Tanzi R.E., MC Clatchey A.I., Lamperti E.D., Villa-Komaroff and Neve R.L. (1988) Nature 331, 528-530.

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Kitagushi N., Takahashi Nature 331, 531-532.

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Weidemann A., Koning G., Bunke D., Fisher and Beyreuther K. (1989) Cell 57, 115-126.

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CL., B. (19871

L., Gusella J.F.,

S., and Ito H. (1988)

P., Salbaum J.M., Masters

C.L.,

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171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

6.

Palmert M.R., Podlisny M.B., Witker D.S., Oltersdorf T., Younkin L.H., Selkoe D. J., and Younkin S.G. (1989) Proc. Natl. Acad. Sci. USA 86, 63386342.

7.

Palmert M., Siedlak S.L., Podlisny M.B., Greenberg B., Shelton E.R., Chan H.W., Usiak M., Selkoe D.J., Perry G., and Younkin S.G. (1990) Biochem. Biophys Res. Commun. 165, 183-188.

8.

Esch F.S., Keim P.S., Beattie E.C., Blather R.W., Culwell A.R., Oltersdorf T., McClure D., and Ward P.J. (1990) Science 248,1122-1124.

9. de Sauvage F., and Octave J.-N. (1989) Science 245, 651-653. 10. Sinha S., Dovey H.F., Seubert P., Ward P.J., Blather R.W., Blaber M., Bradshaw R.A., Arici M., Mobley WC., and Liebrburg I. (1990) J. Biol. Chem. 265, 8983-8985. 11. Edge A.S.B., Faltynek C.R., Hof L., Reichert L.E., and Weber P. (1981) Anal. Biochem. 118, 131-137. 12. Laemmli U.K. (1970) Nature 227, 680-685. 13. Van Nostrand W.E., Wagner S.L., Farrow J.S., and Cunningham D.D. (1990) J. Biol. Chem. 265, 9591-9594.

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